Pro-drug and use thereof as a medicament

ABSTRACT

The invention relates to a pro-drug comprising a cyclic undecapeptide wherein the peptide chain thereof comprises at least one amino acid residue of general formula (1) wherein the carbon atom C a  is one of the links in said undecapeptide cycle; —substituents Y individually represent a hydrogen atom or form a bond together; —substitutents R 1  and R 3  independently represent a hydrogen atom, an aralkyl group, an alkaryl group, a heteroalkyl group, a heterocyclic group, a heterocyclic alkyl group, an alkyl heterocyclic group or an alkyl group having 1-6 carbon atoms which is linear or branched, and which is optionally substituted by at least one of the groups chosen from among —COOH, —CONHR 8 , —NHCO═NH(NH 2 ), —NHC═NR 8 (NH 2 ), —NH 2 , —NHR 8 , —NR 8   2 , —N + R 8   3 , —OH, —OPO(OR 8 ) 2 , —OPO(OH)(OR 8 ), —OPO(OH) 2 , —OSO(OR 8 ) 2 , —OSO(OH)(OR 8 ), —OSO(OH) 2 , and the different salts produced therefrom, each of the substituents R 8  independently representing alkyl group which is linear or ramified having 1-6 carbon atoms; —substituents R 2  and R 4  independently represent a hydrogen atom, an alkaryl group or an alkyl group having 1-6 carbon atoms which is linear or branched; —the substituents R 5  and R 6  individually represent a hydrogen atom, an aralkyl group, an alkyl group having 1-6 carbon atoms which is linear or branched; and substituent R 7  represents an alkaryl group, a heteroalkyl group, a heterocylic group, a heterocylic alkyl group, an alkyl heterocyclic group or an alkyl group having 1-6 carbon atoms which is linear or branched.

[0001] The present invention relates to a pro-drug consisting of acyclic undecapeptide and to the use thereof as a medicinal product,intended in particular for the treatment of pathological conditions ofthe eye.

[0002] Cyclosporins constitute a structurally distinct class of cyclicpeptides which have in common the fact that they consist of a chain ofeleven amino acids, some being atypical either due to their Dconfiguration or due to the complex chemical structure of their sidechain, or else due to the fact that the amine group is alkylated.

[0003] To date, about thirty cyclosporins have been isolated from afungal source and many cyclic undecapeptides similar to these naturalproducts have been obtained by hemisynthesis or by total synthesis. Alsoincluded among these cyclic undecapeptide analogs are peptolides ordepsipeptides, i.e. cyclic polypeptides also containing ester linkagesin their chain.

[0004] In the remainder of this description, and unless otherwisespecified, the term “cyclosporin”will be intended to mean both thecyclic undecapeptides obtained from a natural source and their analogsobtained by hemisynthesis or total synthesis, including the peptolidesobtained from a natural source or their analogues obtained byhemisynthesis or by total synthesis.

[0005] The first member of this cyclosporin family to have been isolatedand then identified was Cyclosporin A. The peptide chain constitutingits undecapeptide ring is as follows:

-MeBmt-Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-

[0006] Its expanded chemical structure being as follows:

[0007] Among the atypical amino acids which this cyclic undecapeptidecomprises, that which is in the 1-position, namelyN-methyl-(4R)-4-((E)-2-butenyl)-4-methyl-L-threonine, called MeBmt, isin particular noted.

[0008] This amino acid is specific to cyclosporins, it being possiblefor the ethylenic group to be optionally reduced. It has an amine groupwhich is methylated. In addition, the hydroxyl group which it carries isvery notable in the sense that it is the only group of this entirecyclic undecapeptide capable of producing chemical modification. It isalso possible to note already that it is in a greatly hindered stearicenvironment making any approach by a reagent extremely delicate.

[0009] These cyclic undecapeptides, whether they are of natural originor are obtained by synthesis, exhibit a broad spectrum of biologicalactivities, among which the most well-known are the imino suppressive,anti-inflammatory or anti-parasitic activities or activities making itpossible to combat or decrease the resistance of cancerous tumors toother treatments. Some of these cyclic undecapeptides have been found topossess promising antiviral activities, in particular in the treatmentof AIDS by inhibition replication of the human immune deficiency virustype 1 (HIV-1).

[0010] In this respect, a certain number of cyclic undecapeptides,obtained by hemisynthesis and having a structure similar to that ofCyclosporin A, but in which the nature of the amino acids in the4-position, or in the 3- and 4-position, with respect to the MeBmt aminoacid has been modified, have been described in patent application WO00/01715 filed by the present applicant.

[0011] Recent pharmacological developments have made it possible to hopethat the immuno moderating effect of cyclosporins, in particular that ofCyclosporin A, which effect is reversible and non-myelotoxic and forwhich few side effects have been listed, may be taken advantage of, inparticular in the field of opthalmology, for local treatment, inparticular, of superficial pathological conditions of the eye and of itssurrounding appendages.

[0012] Included among these pathological conditions are, inter alia, drykeratoconjunctivitis, also called dry eye syndrome, Sjögren's syndrome,forms of allergic keratoconjunctivitis, in particular those resistant tocorticosteroids, conjunctivitis producing mucous and synechia,herpetitic stromal keratitis, immune-related limbic keratitis andThygeson's keratitis, and prevention of corneal transplant rejection,and as an adjuvant treatment for filtering surgery.

[0013] The cyclic undecapeptides of the cyclosporin family are highlyhydrophobic in nature, which reduces all the more their solubility inwater. This characteristic is related to the nature of the side chain ofmost of their amino acids, but also to the fact that the amine group ofsome of these amino acids is methylated, thus limiting the possiblenumber of intermolecular hydrogen bond formations between the cyclicundecapeptide and, for example, an aqueous solubilizing medium.

[0014] As a result, the intravenous (i.v.) administration of thesecyclosporins requires the development of very complex pharmaceuticalformulations, mainly in the form of emulsions, which sometimes haveprecarious stability and are delicate to handle, and which are sourcesof adverse side effects.

[0015] By way of example, one of the preparations for i.v. infusion ofCyclosporin A, commercially available under the trademark Sandimmun,consists of a microemulsion using, as excipient, a polyoxyethylenatedcastor oil known under the trademark Cremophor. This preparation isconserved in the form of a concentrate and must be diluted just beforeit is administered.

[0016] Due to the use of this castor oil, which is known to solubilizesome of the components of synthetic materials, the manufacturerrecommends using, when handling this preparation, only material made ofglass or, failing this, of a synthetic material in accordance with the“standards of the European Pharmacopia for receptacles intended tocontain blood”, all these materials having to be free of silicone oiland of fats.

[0017] In addition, it warns the clinician that this castor oil iscapable of causing anaphylactoid reactions and, as a result, recommendsthat intravenous administration be used only in cases where oraladministration is impossible.

[0018] The development of promising uses of cyclosporins by localadministration in opthalmology remains slow-moving due also to thedifficulty in developing suitable pharmaceutical formulations whichexhibit in particular good local tolerance and do not cause blurredvision due to the presence of viscous agents.

[0019] Thus, and by way of example, Robert et al. have recentlyreviewed, in J. Fr. Opthalmol., 2001, 24(5), 527, all the technicaldifficulties which have to be worked out due to the lypophilic nature ofthe pharmaceutical formulations for administering Cyclosporin A locallyin opthalmology and all the problems of local tolerance which theseformulations cause.

[0020] One of the conclusions which may be drawn from this review isthat, to date, no formulation exists in the form of an eyewash which canbe administered locally for the treatment of conditions of the eye andof its surrounding appendages. This conclusion may be broadened to theuse of cyclosporin for the local treatment of conditions of the mucousmembranes or of skin conditions.

[0021] Consequently, there is still a need to make available toclinicians cyclosporins, whether they are of natural or syntheticorigin, or derivatives of these cyclosporins, which can be made readilyadministerable to a patient, in particular locally or intravenously,while avoiding the use of complex pharmaceutical formulations which havea precarious stability and which are difficult to handle, and which aresources of adverse side effects.

[0022] This need exists all the more if these cyclosporins, of naturalor synthetic origins, must be applied locally to the eye or to itssurrounding appendages.

[0023] One of the possibilities available to the specialist whenconfronted with the problem of making a hydrophobic, pharmacologicallyactive molecule assimilable in a physiological medium is to chemicallymodify it in order to confer on it a hydrophilic nature.

[0024] In order to avoid altering the pharmacological properties of sucha pharmacologically active molecule, this chemical modification mayconsist in preparing a precursor, if possible an inactive precursor, ofthis pharmacologically active molecule, which, once administered andunder the effect of the physiological conditions existing locally in thebody, will be chemically or enzymatically modified such that thepharmacologically active molecule is released, if possible, either atthe site where its pharmacological action must occur, or in the bloodwhich will transport this pharmacologically active molecule thusreleased to its site of action, this corresponding to the “pro-drug”concept. In the remainder of this description, the precursor in questionof said pharmacologically active molecule is called “pro-drug”.

[0025] It is already known practice to chemically modify the structureof Cyclosporin A for the purpose of conferring on the product obtained ahydrophilic nature.

[0026] Thus, Rothbard et al. have described, in patent application WO01/13957, a method for improving the administration of pharmacologicallyactive molecules and for enabling them to cross the dermis and theepithelial membranes, consisting in reversibly grafting onto thesemolecules a side chain consisting of fragments of a polyarginine chain.Included among the pharmacologically active molecules are moleculeswhich are hydrophobic in nature, such as Cyclosporin A.

[0027] However, such conjugates are extremely delicate to handle and toconserve due to the fact, as is indicated in the cited application, thatthe pharmacologically active molecule is released as soon as the pH ofthe medium exceeds 7. In addition, when this pharmacologically activemolecule is released, the polyarginine chain fragments are released inthe body. Since they are known for their toxicity and their irritantcapacity, these polyarginines would cause irritations such that use ofsuch cyclosporin conjugates in the field of opthalmology cannot beenvisioned.

[0028] Crooks et al. describe, in patent application WO 00/67801, thepreparation of pro-drugs of anti-inflammatory agents such asflurbiprofen, in order to be able to administer them locally in contactwith the eye, while at the same time avoiding any local irritation thistime. They achieve this, for a certain number of medicinal products, byintroducing oxygenated or polyoxygenated chains.

[0029] On the other hand, when wishing to subject Cyclosporin A to thesame chemical modifications, they succeeded in obtaining only productswhich they describe as being stable, in other words, which are notcleaved to release the Cyclosporin A, whether this is in contact withhuman serum or in a phosphate buffer at pH 7.4.

[0030] Consequently, the aim of the present invention is to makeavailable to the clinician pro-drugs of cyclic undecapeptides of thecyclosporin family which, firstly, can be administered within thephysiological medium without having to develop complex pharmaceuticalformations and, secondly, can be stored and then handled andadministered without having to worry in particular about the pHconditions of the surrounding medium.

[0031] The aim of the present invention is also to make available to theclinician pro-drugs of cyclic undecapeptides of the cyclosporin familywhich can be administered locally in general, and in particular on thesurface of the eye or on the mucous membranes and which can thenrelease, in a suitable half-life time, the pharmacologically activecyclic undecapeptide, without local irritation.

[0032] To this effect, the present invention relates to a pro-drugconsisting of a cyclic undecapeptide in which the peptide chaincomprises at least one amino acid residue of general formula (I) below:

[0033] in which:

[0034] the carbon atom C^(a) constitutes one of the links of theundecapeptide ring;

[0035] the substituents Y each represent a hydrogen atom or togetherconstitute a bond;

[0036] the substituents R¹ and R³ represent, independently of oneanother, a hydrogen atom, an aralkyl group, an alkaryl group, aheteroalkyl group, a heterocyclic group, an alkylheterocyclic group, aheterocyclicalkyl group or a linear or branched alkyl group having from1 to 6 carbon atoms, said groups being optionally substituted with atleast one of the groups chosen from —COOH, —CONHR⁸, —NHC═NH(NH₂),—NHC═NR⁸ (NH₂), —NH₂, —NHR⁸, —NR⁸ ₂, —N⁺R⁸ ₃, —OH, —OPO(OR⁸)₂,—OPO(OH)(OR⁸), —OPO(OH)₂, —OSO(OR⁸)₂, —OSO(OH)(OR⁸), —OSO(OH)₂, and thevarious salified forms of these groups, each of the substituents R⁸representing, independently of one another, a linear or branched alkylgroup having from 1 to 6 carbon atoms;

[0037] the substituents R² and R⁴ represent, independently of oneanother, a hydrogen atom, an alkaryl group, or a linear or branchedalkyl group having from 1 to 6 carbon atoms;

[0038] the substituents R⁵ and R⁶ represent, independently of oneanother, a hydrogen atom, an aralkyl group, or a linear or branchedalkyl group having from 1 to 6 carbon atoms; and

[0039] the substituent R⁷ represents an aralkyl group, an alkaryl group,a heteroalkyl group, a heterocyclic group, an alkylheterocyclic group, aheterocyclicalkyl group or a linear or branched alkyl group having from1 to 6 carbon atoms, said groups being optionally substituted with atleast one of the groups chosen from —COOH, —CONHR⁸, —NHC═NH(NH₂),—NHC═NR⁸ (NH₂), —NH₂, —NHR⁸, —NR⁸ ₂, —N+R⁸ ₃, —OH, —OPO(OR⁸)₂,—OPO(OH)(OR⁸), —OPO(OH)₂, —OSO(OR⁸)₂, —OSO(OH)(OR⁸), —OSO(OH)₂, and thevarious salified forms of these groups, each of the substituents R⁸having the definition above.

[0040] When the two substituents Y together constitute a bond, saidamino acid residue of general formula (I) derives from anN-methyl-(4R)-4-((E)-2-butenyl)-4-methyl-L-threonine residue in whichthe hydroxyl group of the threonine has been esterified in theappropriate manner, and the pharmacologically active molecule which willbe released when the pro-drug is cleaved in the body will consist of acyclic undecapeptide in which the peptide chain comprises at least oneN-methyl-(4R)-4-((E)-2-butenyl)-4-methyl-L-threonine residue (MeBmt).

[0041] Similarly, when the two substituents Y each represent a hydrogenatom, said amino acid residue of general formula (I) derives from anN-methyl-(4R)-4-butyl-4-methyl-L-threonine residue in which the hydroxylgroup of the threonine has been esterified in the appropriate manner,and the pharmacologically active molecule which will be released whenthe pro-drug is cleaved in the body will consist of a cyclicundecapeptide in which the peptide chain comprises at least oneN-methyl-(4R)-4-butyl)-4-methyl-L-threonine residue (Dh-MeBmt).

[0042] Preferably, in general formula (I) defining said amino acidresidue, at least one of the substituents R¹ and R³ represents anaralkyl group, an alkaryl group, a heteroalkyl group, a heterocyclicgroup, an alkylheterocyclic group, a heterocyclicalkyl group or a linearor branched alkyl group having from 1 to 6 carbon atoms, each of saidgroups being substituted with at least one of the groups chosen from—COOH, —CONHR⁸, —NHC═NH(NH₂), —NHC═NR⁸ (NH₂), —NH₂, —NHR⁸, —NR⁸ ₂, —N⁺R⁸₃, —OH, —OPO(OR⁸)₂ , —OPO (OH)(OR⁸), —OPO(OH)₂, —OSO(OR⁸)₂,—OSO(OH)(OR⁸), —OSO(OH)₂, and the various salified forms of thesegroups, each of the substituents R⁸ having the definition above. Thesegroups, acknowledged to be polar in nature, greatly improved thehydrophilic nature conferred on said pro-drug.

[0043] More preferably, said aralkyl, alkaryl, heteroalkyl,heterocyclic, alkylheterocyclic, heterocyclicalkyl or alkyl groups aboveare substituted with at least one of the groups chosen from —NR⁸ ₂,—N⁺R⁸ ₃, —OPO(OH)₂ or the various salified forms of these groups, eachof the substituents R⁸ having the definition above.

[0044] More preferably, at least one of said substituents R¹ and R³represents a linear alkyl group having from 1 to 6 carbon atomssubstituted with at least one of the groups chosen from —NR⁸ ₂, —N⁺R⁸ ₃,—OPO(OH)₂ or the various salified forms of these groups, each of thesubstituents R⁸ having the definition above.

[0045] When said substituents R¹ and R³ represent a linear alkyl grouphaving from 1 to 6 carbon atoms substituted with at least one of thegroups chosen from —NR⁸ ₂, —N⁺R⁸ ₃, —OPO(OH)₂ or the various salifiedforms of these groups, each of the substituents R⁸ having the definitionabove, the corresponding amino acid residues preferably derive:

[0046] either from serine, homoserine, threonine, allothreonine,N-methylserine, N-methylthreonine or N-methylhomoserine residues, in anyone of the (D) or (L) configurations, preferably the (L) confirmation,and in which the hydroxyl group has been functionalized in theappropriate manner such that the side chain of these amino acid residuescarries the polar and/or solubilizing groups;

[0047] or from lysine, ornithine, arginine, N-delta-methylarginine,N-alpha-methylarginine or N-methyllysine residues, in any one of the (D)or (L) configurations, preferably the (L) configuration, and in whichthe respectively amine or imine group has been functionalized in theappropriate manner such that the side chain of these amino acid residuescarries the polar and/or solubilizing groups.

[0048] When the substituents R¹, R² and/or R³, and R⁴ forming the pairs(R¹, R²) and/or (R³, R⁴) are alkyl groups having from 1 to 6 carbonatoms, they can form, within each pair, an alkylene chain which forms,with the carbon atom and the nitrogen atom which carry them, a ring.Preferably, they constitute the side chain of a proline residue.

[0049] When the substituents R¹ and R³ represent, independently of oneanother, a hydrogen atom, an aralkyl group, an alkaryl group, aheteroalkyl group, a heterocyclic group, an alkylheterocyclic group, aheterocyclicalkyl group or a linear or branched alkyl group having from1 to 6 carbon atoms, but said groups are not substituted with at leastone of the groups chosen from —COOH, —CONHR⁸, —NHC═NH(NH₂), —NHC═NR⁸(NH₂), —NH₂, —NHR⁸, —NR⁸ ₂, —N⁺R⁸ ₃, —OH, —OPO(OR⁸)₂, —OPO(OH)(OR⁸),—OPO(OH)₂, —OSO(OR⁸)₂, —OSO(OH)(OR⁸), —OSO(OH)₂, and the varioussalified forms of these groups, then they preferably represent the sidechains of amino acid residues, in (D) or (L) configurations, preferablyin the (L) configuration, or of residues of said amino acids inprotected and/or activated forms and optionally having their amine groupalkylated, which are usually commercially available. More preferably,said amino acid residues are chosen from the twenty amino acids usuallycalled natural amino acids.

[0050] Also preferably, in general formula (I), the substituents R⁵ andR⁶ cannot simultaneously represent a hydrogen atom. Also preferably, atleast one of said substituents R⁵ and R⁶ represents a linear or branchedalkyl group having from 1 to 6 carbon atoms, and the substituent R⁷represents an aralkyl group or a linear or branched alkyl group havingfrom 1 to 6 carbon atoms.

[0051] More preferably, said substituents R⁵ and R⁶ represent,independently of one another, a hydrogen atom or a methyl group.

[0052] Preferably, said pro-drug consists of a cyclic undecapeptide inwhich the peptide chain comprises a single amino acid residue of generalformula (I) and thus forms an undecapeptide ring with a linear sequenceof ten amino acids of general formula (II) below:

-T-U-V-W-MeLeu-Ala-X-MeLeu-Z-MeVal-  (II)

[0053] in which:

[0054] T is chosen from the amino acids Ala, Abu, Nval, Val and Thr;

[0055] U is chosen from the amino acids Sar, (D)MeSer, (D)MeAla and(D)MeSer(OCOR⁹), with R⁹ representing a hydrogen atom, an alkaryl group,or a linear or branched alkyl group having from 1 to 6 carbon atoms;

[0056] V represents an amino acid of the general formula (N-R¹⁰)aa, aabeing chosen from the amino acids Val, Leu, Ile, Thr, Phe, Tyr and Thrand R¹⁰ being a linear or branched alkyl group having from 1 to 6 carbonatoms;

[0057] W is chosen from the amino acids Val, Nval and Leu;

[0058] X is chosen from the amino acids (D)Ala, (D)ser, (D)Hiv, (D)Valand (D)Thr, with (D)Hiv representing a D-2-hydroxyisovaleric acidresidue; and

[0059] Z is chosen from the amino acids Leu and MeLeu.

[0060] Thus, when, in said amino acid residue of general formula (I),the two substituents Y each represent a hydrogen atom, thepharmacologically active molecule which will be released, during thecleavage of the pro-drug in the body, will consist of a cyclicundecapeptide of the cyclosporin family in which the peptide chaincontains an N-methyl-(4R)-4-butyl-4-methyl-L-threonine residue(Dh-MeBmt).

[0061] Similarly, when, in said amino acid residue of general formula(I), the two substituents Y together constitute a bond, thepharmacologically active molecule which will be released, during thecleavage of the pro-drug in the body, will consist of a cyclicundecapeptide of the cyclosporin family in which the peptide chaincomprises an N-methyl-(4R)-4-((E)-2-butenyl)-4-methyl-L-threonine(MeBmt) residue.

[0062] Preferably, these cyclic undecapeptides correspond to thecyclosporins already described in the literature as havingpharmacological properties, and all having, in their peptide chain,either an N-methyl-(4R)-4-((E)-2-butenyl)-4-methyl-L-threonine (MeBmt)residue or an N-methyl-(4R)-4-butyl-4-methyl-L-threonine residue(Dh-MeBmt).

[0063] More preferably, the linear sequence of the ten remaining aminoacid residues constituting, with said amino acid residue of generalformula (I), said cyclic undecapeptide is chosen from the followingsequences of formulae (III) to (XIV):

-Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (III);

-Abu-(D)MeAla-EtVal-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (IV);

-Thr-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (V);

-Val-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (VI);

-Nval-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (VII);

-Val-(D)MeAla-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (VIII);

-Val-Sar-MeLeu-Val-MeLeu-Ala-(D)Val-MeLeu-Leu-MeVal-  (IX);

-Val-Sar-MeLeu-Val-MeLeu-Ala-(D)Thr-MeLeu-Leu-MeVal-  (X);

-Abu-(D)MeSer(OAc)-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-Leu-MeVal-  (XI);

-Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ser-MeLeu-MeLeu-MeVal-  (XII);

-Thr-Sar-MeLeu-Leu-MeLeu-Ala-(D)-Hiv-MeLeu-Leu-MeVal-  (XIII);

and -Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)Val-MeLeu-Leu-MeVal-  (XIV).

[0064] The pharmacologically active molecule which will be releasedduring the cleavage of the pro-drug in the body will then berespectively one of the following cyclosporins with, as appropriate, aresidue derived from threonine with a butenyl (MeBmt) or butyl(Dh-MeBmt) chain:

[0065] Cyclosporin A (CsA); (D)MeAla³EtVal⁴CsA (WO 00/01715);Cyclosporin C (CsC); Cyclosporin D (CsD); Cyclosporin G (CsG);(D)MeAla³CsD; (D)Val⁸Csl; (D)Thr⁸Csl; (D)MeSer(Oac)³CsT; (D)Ser⁸CsA(Progress in Medicinal Chemistry, Vol 25, ed. Ellis and West, ElsevierScience Publ., Biomedical Division, 1998, pp 1-33);Thr²Leu⁵(D)Hiv⁸Leu¹⁰CsC (The Journal of Biological Chemistry, 1991,266(24), 15570); (D)Val⁸Leu¹⁰CsA; cyclosporins A, C, D, G, I and T beingdescribed in Progress in the Chemistry of Organic Natural Products,1986, 50, 124, the remaining cyclosporins being prepared by analogy withthe method described in Helvetica Chimica Acta, 1984, 67, 502.

[0066] More preferably, the pro-drugs of the present invention have,respectively, formulae (XV) and (XVI) below:

[0067] The pro-drugs according to the present invention can be preparedby applying methods of chemical synthesis well known to the specialistin peptide chemistry, and most particularly in cyclosporin chemistry.

[0068] By virtue of an appropriate choice of the various substituentsdefining the amino acid residue of general formula (I), the pro-drugsaccording to the present invention have been found to have, notably, agreatly enhanced hydrophilic nature compared to the pharmacologicallyactive molecule generated during the cleavage of said pro-drug. By wayof example, the solubility of certain pro-drugs of the presentinvention, which generate Cyclosporin A after cleavage, is at least 3000times greater than that of Cyclosporin A.

[0069] Consequently, the pro-drugs of the present invention can beeasily incorporated into aqueous pharmaceutical formulation.

[0070] Also notably, the pro-drugs of the present invention are foundnot to be sensitive to the pH conditions usually encountered for thistype of application when they are in aqueous solution.

[0071] In addition, the pro-drugs of the present invention completelyfulfill their role by releasing, with a half-life time entirely suitablefor a therapeutic application, the pharmacologically active moleculewhen they are in contact with the enzymes present in the biologicalhumors.

[0072] The present invention also relates to the use of a pro-drug asdescribed above, as a medicinal product.

[0073] Such a medicinal product is preferably used for the treatment ofpathological conditions or physiological conditions requiring beforehandthe use of a cyclosporin, in particular of all pathological conditionsrequiring the use of Cyclosporin A, locally or systemically byintravenous injection.

[0074] Such a medicinal product is in particular intended to allowprolonged survival of allografts of organs such as the kidney, heart,liver, pancreas, lung, small intestine or bone marrow. It may also beintended to inhibit replication of the human immunodeficiency virus type1 (HIV-1).

[0075] In such applications, the dosage of the pro-drug of the presentinvention when administered systemically by intravenous injection issuch that the concentration of cyclosporin generated during cleavage,for example of Cyclosporin A corresponds to the therapeuticconcentrations usually recommended.

[0076] More preferably, such a medicinal product is used in the field ofophthalmology and is intended, in particular, for the treatment ofpathological conditions of the eye and of its surrounding appendages.

[0077] Included among these pathological conditions are, inter alia, drykeratoconjunctivitis, also called dry eye syndrome, Sjögren's syndrome,forms of allergic keratoconjunctivitis, in particular those resistant tocorticosteroids, conjunctivitis producing mucous and synechia, herpeticstromal keratitis, immune-related limbic keratitis and Thygeson'skeratitis, and prevention of corneal transplant rejection, and as anadjuvant treatment for filtering surgery. More preferably, the medicinalproduct of the present invention is used for the treatment of drykeratoconjunctivitis.

[0078] In such applications, the dosage of the pro-drug of the presentinvention is such that the lachrymal concentration of cyclosporingenerated during cleavage of the pro-drug, for example of Cyclosporin A,should be greater than 0.5 μg/l by local administration.

[0079] The medicinal product of the present invention can beadministered topically, in particular for the local treatment ofconditions of the mucous membranes or of skin conditions, orparenterally, in particular intravenously. It may also be administeredorally, with the aim of improving the bioavailability of thecyclosporin, for example of the Cyclosporin A.

[0080] When the medicinal product of the present invention isadministered parenterally, suitable pharmaceutical preparations may besterile, concentrated aqueous solutions, or powders for injectablepreparations.

[0081] Preferably, the medicinal product of the present invention isadministered intravenously. Suitable pharmaceutical preparations forsuch an administration are aqueous solutions for injection or forinfusion which are well known to the specialist.

[0082] More preferably, the medicinal product of the present inventionis administered locally. Suitable pharmaceutical preparations for suchan administration, in particular for an ophthalmic application, are eyewashes, in the form of aqueous sterile solutions, ophthalmic ointments,ophthalmic gels and ophthalmic inserts.

[0083] The present invention, and also its advantageous properties, arepresented in detail, without, however, being limiting, in the examplesand with the aid of the drawing in which,

[0084]FIG. 1 represents a curve of in vitro conversion kinetics for apro-drug according to the invention by hydrolysis with esterases and acurve of the kinetics of appearance of Cyclosporin A;

[0085]FIG. 2a represents the Cyclosporin A level in the blood after i.v.administration of Cyclosporin A in an oily form in rats;

[0086]FIGS. 2b and 2 c represent the Cyclosporin A level in the bloodafter i.v. administration of, respectively, an aqueous solution of twoof the pro-drugs according to the invention in rats; and

[0087]FIG. 3 represents, respectively, the concentrations over time ofCyclosporin A and of a pro-drug according to the invention, in rabbittears.

[0088] In the nomenclature used in the examples to describe the productsobtained, the Cyclosporin A residue will be designated by theabbreviation—CsA, the residue of the opposite fragment being linked tothe only functionalizable group of this cyclic undecapeptide, namely thehydroxyl group of the amino acid having the 1-position,N-methyl-(4R)-4-((E)-2-butenyl)-4-methyl-L-threonine (MeBmt). Thestructural chemical formulae of the intermediate products derived fromthe Cyclosporin A will represent only the amino acid residue in the1-position with the respective side chain.

EXAMPLE 1 Preparation of the Cyclic Undecapeptide of Formula (XV)

[0089]

1. Preparation of MeBmt(O-Sar-Lys(N_(ε) ⁺Me₃)-COOCH(CH₃)OCOCH₃))¹-CsA(XV)

[0090] 1.1. Preparation of α-acetoxyethyl para-nitrophenyl Carbonate (2)

[0091] 1.1.1. Preparation of α-chloroethyl para-nitrophenyl Carbonate(1)

[0092] 2.6 ml (23.7 mmol, 1.1 eq) of α-chloroethyl chloroformate areadded, at 0° C., to a solution of 3 g (21.6 mmol, 1 eq) of p-nitrophenoland 1.7 ml (21.7 mmol, 1 eq) of pyridine in 108 ml of chloroform. Thereaction mixture is stirred for 30 min at 0° C. and then for 16 h atambient temperature. The reaction mixture is extracted with water, witha 0.5% NaOH solution and then with water. The organic phase is driedover Na₂SO₄, filtered and evaporated under reduced vacuum, to give ayellow oil which gives a pure white solid (5.8 g) after crystallizationfrom hexane.

[0093]¹H-NMR (400 MHz, CDCl₃); δ: 8.32 (d, 2H), 7.44 (d, 2H), 6.52 (q,1H), 1.95 (s, 3H).

[0094] 1.1.2. Preparation of α-acetoxyethyl para-nitrophenyl Carbonate(2)

[0095] 7.8 g (24.4 mmol, 1.5 eq) of mercury acetate are added to asolution of 4 g (16.3 mmol, 1 eq) of (1) dissolved in 100 ml of aceticacid. The reaction mixture is stirred for 1 day at ambient temperature,and then an additional 1 g (3.13 mmol, 0.2 eq) of mercury acetate isadded. After stirring for a further 1 day at ambient temperature, thereaction is complete. The acetic acid is evaporated off under highvacuum and the residue is taken up in ether. The organic phase isextracted with a saline solution and then dried over Na₂SO₄, filteredand evaporated under reduced vacuum, to give a yellow oil. The crudeproduct is chromatographed on silica gel, to give a colorless oil (4.4g).

[0096]¹H-NMR (400 MHz, CDCl₃); δ: 8.31 (d, 2H), 7.43 (d, 2H), 6.87 (q,1H), 2.16 (s, 3H), 1.64 (s, 3H).

[0097] 1.2. Preparation of α-acetoxyethoxycarbonyl-lysine (N_(ε)(Fmoc))(5)

[0098] 1.2.1. Preparation of the Benzyl Ester ofα-acetoxyethoxycarbonyl-lysine (N_(ε)(Z)) (3)

[0099] 500 mg (1.23 mmol, 1 eq) of H-Lys(Z)Obn.HCl are suspended in 2.5ml of dioxane. 231 μl (1.35 mmol, 1.1 eq) of N,N-diisopropylethylamine(DIPEA) and 396 mg (1.47 mmol, 1.2 eq) of (2) are added at ambienttemperature. After stirring for 1 day at ambient temperature, thereaction is complete. The dioxane is evaporated off under reduced vacuumand the residue is taken up in 20 ml of ethyl acetate. The organic phaseis extracted three times with a 6% citric acid solution (20 ml), asaturated NaHCO₃ solution (20 ml) and a saturated NaCl solution (20 ml),dried over anhydrous Na₂SO₄ and filtered, and the solvent is evaporatedoff under reduced vacuum. The crude product obtained is chromatographedon silica gel, to give a clear, transparent oil (576 mg).

[0100] ESI-MS: m/z: 501.34 [M+H⁺]; 518.28 [M+H₂O+H⁺].

[0101] 1.2.2. Preparation of α-acetoxyethoxycarbonyl-lysine (4)

[0102] 50 mg of palladium on active charcoal are added to a solution of509 mg (1.02 mmol) of (3) in 10 ml of ethanol. After stirring for 3 h atambient temperature under a stream of hydrogen, the reaction iscomplete. The reaction mixture is filtered over celite and the filtrateis evaporated under reduced vacuum, to give the crude in the form ofbrownish crystals, which is used directly in the following step (254mg).

[0103] ES-MS m/z: 276.87 [M+H⁺].

[0104] 1.2.3 Preparation of α-acetoxyethoxycarbonyl-lysine (N_(ε)(Fmoc))(5)

[0105] 1.11 ml of DIPEA (6.53 mmol, 1.7 eq) and 1.555 g (4.61 mmol, 1.2eq) of Fmoc-O-Suc are added to a solution of 1.062 [lacuna] (3.84 mmol,1 eq) of (4) dissolved in 38 ml of dioxane. After stirring for 1 h atambient temperature, the reaction is complete. The dioxane is evaporatedoff under reduced vacuum and the residue is taken up in 20 ml of EtOAc.The organic phase is washed once with a 6% citric acid solution (20 ml)and a saturated NaCl solution (20 ml), dried over anhydrous Na₂SO₄ andfiltered, and the solvent is evaporated off under reduced vacuum. Thecrude product obtained is chromatographed on silica gel, to give a whitefoam (1.026 g).

[0106] ES-MS m/z: 499.37 [M+H⁺], 516.29 [M+H₂O+H⁺].

[0107] 1.3. Preparation of MeBmt(O-Sar-Lys((N_(ε)⁺Me₃)-COOCH(CH₃)OCOCH₃))¹-CsA.I⁻ (XV)

[0108] 1.3.1. Preparation of MeBmt(O-COCH₂Br)¹-CsA (6)

[0109] 4 g (3.33 mmol, 1 eq) of dry CsA are dissolved under argon in 66ml (0.76 mol) of bromoacetyl bromide. 2 g (16.64 mmol, 5 eq) ofdimethylaminopyridine are added in small portions, and the reactionmixture is stirred at ambient temperature for 40 min. The reaction isthen complete. The reaction mixture is poured, carefully and withvigorous stirring, into a mixture of hydrogen carbonate (77 g, 0.91mol), water (500 ml) and crushed ice. The possible addition of a fewfurther portions of NaHCO₃ makes it possible to bring the pH of thesolution to 7-8. The separated aqueous phase is extracted twice withdichloromethane and the pooled organic phases are extracted three timeswith a saturated NaHCO₃ solution and with a saturated NaCl solution,dried over anhydrous Na₂SO₄ and filtered, and the solvent is evaporatedoff under reduced vacuum. The crude product obtained is chromatographedon silica gel, to give a white foam (3.2 g).

[0110] ESI-MS: m/z: 622.6 [M+2H⁺], 673.9 [M+Na⁺+H⁺].

[0111] 1.3.2. Preparation of MeBmt(O-Sar-H)¹-CsA (7)

[0112] 300 mg (0.23 mmol, 1 eq) of (6) are dissolved in 2.3 ml ofethanol. 95 μl (0.68 mmol, 3 eq) of triethylamine (TEA) and 31 mg (0.45mmol, 2 eq) of additional methylammonium chloride are added at ambienttemperature. After stirring for 3 days at ambient temperature,adjustment of the pH to 12 by adding TEA and addition of 15 mg (0.23mmol, 1 eq) of methylammonium chloride, the reaction is complete after 1hour. The ethanol is evaporated off under reduced vacuum and the residueis taken up in ethyl acetate. The organic phase is extracted with waterand with a saturated NaCl solution, dried over anhydrous Na₂SO₄ andfiltered, and the solvent is evaporated off under reduced vacuum. Thecrude product obtained is chromatographed on silica gel, to give a whitefoam (200 mg).

[0113] ESI-MS: m/z: 1273.7 [M+H⁺].

[0114] 1.3.3. Preparation ofMeBmt(O-Sar-Lys(N_(ε)(FMOC-COOCH(CH₃)OCOCH₃))¹-CsA (8)

[0115] 79 mg (0.06 mmol, 1 eq) of (7) are dissolved in 0.5 ml ofdichloromethane (DCM) under argon. 31.6 μl (0.18 mmol, 3 eq) of DIPEA,35 mg (0.09 mmol, 1.5 eq) ofO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) and 40 mg (0.08 mmol, 1.3 eq) of (5)dissolved in 0.8 ml of DCM are added successively under argon. Afterstirring for 3 h at ambient temperature, the reaction is complete. TheDCM is evaporated off under reduced vacuum and the residue is taken upin 20 ml of EtOAc. The organic phase is washed once with a 6% citricacid solution (20 ml), a saturated NaHCO₃ solution (20 ml) and asaturated NaCl solution (20 ml), dried over anhydrous Na₂SO₄ andfiltered, and the solvent is evaporated off under reduced vacuum. Thecrude product is chromatographed on silica gel, to give a white foam (59mg).

[0116] ES-MS m/z: 1754.36 [M+H⁺]; 877.86 [M+2H⁺].

[0117] 1.3.4. Preparation ofMeBmt(O-Sar-Lys(N_(α)-COOCH(CH₃)OCOCH₃))¹-CsA (9)

[0118] 90 μl (0.86 mmol, 10 eq) of diethylamine are added to a solutionof 150 mg (0.09 mmol, 1 eq) of (8) dissolved in 900 μl of acetonitrile.After stirring for 3 h at ambient temperature, the reaction is complete.The solvent is evaporated off under reduced vacuum and the crude productis chromatographed on silica gel, to give a white foam (52 mg).

[0119] ES-MS m/z: 1532.79 [M+H⁺]; 766.79 [M+2H⁺].

[0120] 1.3.4. Preparation of MeBmt(O-Sar-Lys(N_(ε)⁺Me₃)-COOCH(CH₃)OCOCH₃))¹-CsA.I⁻ (XV)

[0121] 49 mg (0.03 mmol, 1 eq) of (9) are dissolved in 640 μl ofanhydrous DCM, and then 30 μl (0.48 mmol, 15 eq) of MeI are added,followed by 14 μl (0.08 mmol, 2.5 eq) of DIPEA. After stirring for 1hour at ambient temperature, the reaction is complete. The DCM isevaporated off under reduced vacuum and the crude product is purified bysemi-preparative HPLC in order to isolate the pure compound in the formof a lyophilisate (30 mg).

[0122] ES-MS m/z: 1574.37 [M+H⁺], 787.83 [M+2H⁺].

EXAMPLE 2 Preparation of the Cyclic Undecapeptide of Formula (XVI)

[0123]

1. Preparation of MeBmt(O-Sar-Ser(OPO(OH₂))-COOCH(CH₃)OCOCH₃)¹-CsA,(XVI)

[0124] 1.1. Preparation of α-acetoxyethoxycarbonyl-serine (11)

[0125] 1.1.1. Preparation of the Benzyl Ester ofα-acetoxyethoxycarbonyl-serine (10)

[0126] 1.4 g (6.04 mmol, 1 eq) of H-Ser-OBn.HCl are suspended in 12 mlof dioxane. 1.14 ml (6.64 mmol, 1.1 eq) of DIEA and 2.1 g (7.85 mmol,1.3 eq) of (2) obtained according to Example 1 are added at ambienttemperature. After stirring overnight at ambient temperature, thereaction is complete. The dioxane is evaporated off under reducedpressure and the residue is taken up in ethyl acetate. The organic phaseis extracted three times with a 6% citric acid solution, a saturatedNaHCO₃ solution and a saturated NaCl solution, dried over anhydrousNa₂SO₄ and filtered, and the solvent is evaporated off under reducedvacuum. The crude product obtained is chromatographed on silica gel, togive a white foam (1.7 g).

[0127]¹NMR (400 MHz, CDCl₃): δ: 7.34-7.41 (m, 5H), 6.79-6.85 (m, 1H),5.72-5.82 (m, 1H), 5.24 (s, 2H), 4.47 (m, 1H), 3.94-4.05 (m, 2H), 2.06and 2.08 (s, 3H), 1.49 and 1.50 (d, 3H).

[0128] 1.1.2. Preparation of α-acetoxyethoxycarbonyl-serine (11)

[0129] 40 mg of palladium on active charcoal are added to a solution of400 mg (1.23 mmol) of (10) in 13 ml of ethanol. After stirring for 4 hat ambient temperature under a stream of hydrogen, the reaction iscomplete. The reaction mixture is filtered over celite and the filtrateis evaporated under reduced vacuum to give the crude in the form of atranslucent deposit, which is directly used in the following step (328mg).

[0130]¹NMR (400 MHz, CDCl₃): δ: 6.3-6.8 (m, 1H), 5.8-6.3 (m, 1H),4.3-4.4 (m, 1H), 3.7-4.1(m, 2H), 2.08 (s), 1.49 and 1.50 (d, 3H).

[0131] 1.2. Preparation ofMeBmt(O-Sar-Ser(OPO(OH)₂)-COOCH(CH₃)OCOCH₃)¹-CsA, (XVI)

[0132] 1.2.1. Preparation ofMeBmt(O-Sar-Ser(OPO(OH)₂)-COOCH(CH₃)OCOCH₃)¹-CsA, (12)

[0133] 170 mg (0.13 mmol) of (7) obtained according to Example 1 aredissolved in 3 ml of dichloromethane under argon. 92 μl (0.53 mmol, 4eq) of DIEA, 51 mg (0.26 mmol, 2 eq) of HATU and 60 mg of (11) (0.25mmol, 2 eq) are added successively under argon. After stirring for 5hours at ambient temperature, the reaction is complete. Thedichloromethane is evaporated off under reduced vacuum and the residueis taken up in ethyl acetate. The organic phase is extracted three timeswith a 6% citric acid solution, a saturated NaHCO₃ solution and asaturated NaCl solution, dried over anhydrous Na₂SO₄ and filtered, andthe solvent is evaporated off under reduced vacuum. The crude productobtained is chromatographed on silica gel, to give a white foam (143mg).

[0134] ESI-MS: M/z: 1513.32 [M+Na⁺], 1508.34 [M+H₂O+H⁺), 1491.36 (M+H⁺],746.36 [M+2H⁺]

[0135] 1.2.2. Preparation ofMeBmt(O-Sar-Ser(OPO(Oall)₂)-COOCH(CH₃)OCOCH₃)¹-CsA (13)

[0136] 130 mg (0.09 mmol, 1 eq) of (12) are dissolved in 880 μl ofanhydrous CH₂Cl₂. 20 mg (0.27 mmol, 3 eq) of 1H-tetrazole are thenadded, followed by 52 μl (0.17 mmol, 2 eq) of (AllO)₂PN(iPr)₂. Afterstirring for 4 h at ambient temperature, the reaction mixture is cooledto −60° C., 44 mg (0.17 mmol, 2 eq) of m-chloroperbenzoic acid areadded, and the stirring is continued for 30 min at −60° C., 15 min at 0°C., and 45 min at ambient temperature. 0.5 ml of a 10% Na₂S₂O₅ solutionare added, at 0° C., to the reaction medium in order to destroy theexcess oxidant, and then extraction is carried out with dichloromethane.The organic phase is washed with a 10% Na₂S₂O₅ solution and thedichloromethane is then evaporated off under reduced vacuum. The residueis taken up in methyl tert-butyl ether and this organic phase isextracted with a 6% citric acid solution and a saturated NaCl solution,dried over anhydrous Na₂SO₄ and filtered, and the solvent is evaporatedoff under reduced vacuum. The crude product obtained is chromatographedon silica gel, to give a white foam (98 mg).

[0137] ESI-MS: m/z: 1651.38 [M+H⁺], 826.35 [M+2H⁺].

[0138] 1.2.3. Preparation ofMeBmt(O-Sar-Ser(OPO(OH)₂)-COOCH(CH₃)OCOCH₃)¹-CsA (XVI)

[0139] 206 μl (1.55 mmol, 8 eq) of Me₃SiN₃ and 1.12 g (0.97 mmol, 5 eq)of (PPh₃)₄Pd° are added, under argon and at ambient temperature, to asolution of 184 mg (0.58 mmol, 3 eq) of Bu₄N⁺F⁻H₂O in 2 ml of CH₂Cl₂.After stirring at ambient temperature for ten minutes, 320 mg (0.19mmol, 1 eq) of (13) are added and the reaction mixture is left to stirat ambient temperature for thirty minutes. The reaction is thencomplete. The reaction mixture is hydrolyzed by adding a 6% citric acidsolution and the dichloromethane is evaporated off under reduced vacuum.The residue is taken up in ethyl acetate and the organic phase obtainedis extracted three times with a 6% citric acid solution and a saturatedNaCl solution, dried over anhydrous Na₂SO₄ and filtered, and the solventis evaporated off under reduced vacuum. The crude product obtained ischromatographed on a Sep-Pack® cartridge and then on preparative HPLC,in order to isolate the pure compound in the form of a lyophilisate (342mg).

[0140] ESI-MS: m/z: 1593.32 [M+Na⁺], 1571.81 [M+H⁺].

EXAMPLE 3 Physicochemical Properties of the Cyclic Undecapeptides ofFormulae (XV) and (XVI) 1. Water-solubility of the Cyclic Undecapeptidesof the Formula e(XV) and (XVI)

[0141] The water-solubilities were evaluated by visual examination atthe ambient temperature of the laboratory, by directly dissolving aweighed amount of cyclic undecapeptide in 67 mM phosphate buffers of theSorensen type. The values are given in Table 1. TABLE 1 Water-solubilityat Cyclic undecapeptide pH = 5 pH = 6 pH = 7 pH = 8 (XV)  >7 mM(XVI) >20 mM >20 mM >20 mM >20 mM

[0142] By way of indication, Cyclosporin A is described as having amaximum water-solubility of 33 μg/ml at a temperature of 20° C. and at apH 7, which corresponds to a maximum concentration of 0.027 mM.

2. Chemical and Enzymatic Stability of the Cyclic Undecapeptide Formulae(XV) and (XVI)

[0143] A first evaluation of the chemical stability over time of thecyclic undecapeptide of formula (XV) was carried out, firstly in anisotonic solution consisting of mannitol and, secondly, in a phosphatebuffer (PBS) at a pH of 7 and at temperature of 4, 20 and 37° C.

[0144] The percentages of Cyclosporin A detected are given in Table 2below: TABLE 2 4° C. 20° C. 37° mannitol mannitol mannitol 37° C. PBS 30days 3.5% 17.0% 25.0% 90.0% 90 days 5.0% 38.0%

[0145] As can be noted, at the temperature of 4° C. in a solution ofmannitol, the cyclic peptide proves to be stable to respond for at least90 days.

[0146] A second study of stabilities, both chemical and enzymatic, wascarried out with the two cyclic undecapeptides of formulae (XV) and(XVI), dissolved in a 50 mM Hepes buffer at pH 7.4, at 37° C., in thepresence and absence of esterases. During the incubation at 37° C., 40μl aliquots are sampled at appropriate time periods and analyzed by HPLCand ESI-MS.

[0147] In the absence of enzyme, a chemical stability of greater than 3days is observed for the two cyclic undecapeptides.

[0148] The results obtained during the hydrolysis in the presence ofesterases have been given in FIG. 1. The curve of conversion kineticsfor the cyclic undecapeptide (XVI) is represented by diamonds, while thecurve of kinetics of appearance of Cyclosporin A is represented bysquares. As can be noted, according to this figure, in the presence ofesterases, the cyclic undecapeptide (XVI) rapidly degrades to releasethe Cyclosporin A. Similar observation was made for the cyclicundecapeptide (XV).

[0149] The cyclic undecapeptide (XV) and (XVI) were subjected toincubation in bovine serum at 37° C. The half-life times of conversionof the cyclic undecapeptides to Cyclosporin A were evaluated and are3.66 and 3.50 hours, respectively.

EXAMPLE 4 Application for Intravenous Administration in the Form of anAqueous Solution Study of Pharmacokinetics of the Cyclic Undecapeptidesof Formulae (XV) and (XVI)

[0150] In order to make available to the clinician a tool which is analternative to the pharmaceutical formulations of Cyclosporin A commonlyused in the form of a microemulsion in polyoxyethylenated castor oil,which has a delicate stability, is relatively difficult to handle andinduces adverse effects, pro-drugs of the present invention, in the formof simple aqueous solutions, were evaluated for the purpose ofintravenous application.

[0151] Thus, the two cyclic undecapeptides (XV) and (XVI), in solutionin phosphate buffer, were administered intravenously to rats at a doseequivalent to 10 mg/kg of Cyclosporin A.

[0152] As a reference, a sample of an injectable solution ofCyclosporin, commercially available on the trademark Sandimmun, was usedafter suitable dilution.

[0153] Blood samples were taken at regular intervals and then subjectedto analysis for the purpose of assaying the Cyclosporin A.

[0154] The Cyclosporin A levels in the blood after i.v. administrationof Cyclosporin A have been given in FIG. 2a. Those obtained after i.v.administration of, respectively, an aqueous solution of the twopro-drugs (XV) and (XVI) are given in FIGS. 2b and 2 c, respectively.The results interpreted from the curves in FIGS. 2a, 2 b and 2 c havebeen given in Table 3 below. TABLE 3 Undecapeptide Cyclosporin A (XV)(XVI) AUC_(0-inf) μg · 69229 66626 44699 h · L⁻¹ CL L/h/kg 0.14 0.150.22 MRT h 15.2 15.3 14.9 Vss L/kg 2.2 2.2 3.4 T1/2₁h 0.31 0.55 0.20T1/2₂h 11.1 11.8 11.8

[0155] In this table, the abbreviations have the following meanings:

[0156] AUC: area under the curve;

[0157] CL: clearance;

[0158] MRT: mean residence time;

[0159] Vss: volume of distribution at steady state;

[0160] T1/2₁: initial half-life time; and

[0161] T1/2₂: terminal half-life time.

[0162] As can be noted from FIG. 2 and from Table 3, the pharmacokineticparameters of the control experiment with Cyclosporin A are comparableto those reported in the literature. The area under the curve forCyclosporin A generated during cleavage of the cyclic undecapeptide (XV)is comparable to that of the Cyclosporin A in the control experiment,whereas that of the Cyclosporin A generated during cleavage of thecyclic undecapeptide (XVI) is decreased by 25% compared to that of theCyclosporin A of the control experiment.

[0163] The two cyclic undecapeptides (XV) and (XVI) each show a similarblood release profile for Cyclosporin A. These profiles are similar tothat of the Cyclosporin A in the pharmaceutical form based onpolyoxyethylenated castor oil.

[0164] From these results, it results that the pro-drugs of the presentinvention offer, for an equivalent Cyclosporin A release profile, thefollowing considerable advantages compared to the existingpharmaceutical formulations of Cyclosporin A:

[0165] easy to use by simply dissolving in water; and

[0166] no need to use excipients which prove to be toxic; and

[0167] no need to use specific materials for handling them.

EXAMPLE 5 Application for Topical Administration to the Eye in the Formof an Aqueous Solution Study of Pharmacokinetics of the CyclicUndecapeptide of the Formula (XV)

[0168] In order to make available to the clinician a tool for topicaladministration of Cyclosporin A to the eye without irritation or anunpleasant sensation being felt or without blurred vision beingexperienced, pro-drugs of the present invention, in the form of simpleaqueous solutions were evaluated.

[0169] 1. Preparation of Solutions

[0170] Isotonic aqueous solutions, containing 5% of mannitol and at pH7.0, of the cyclic undecapeptide (XV) were prepared. The concentrationof Cyclosporin A equivalent is 1% (weight/volume). The solutions weresterilized by passing them through 0.22 μm nitrocellulose filters. Areference formulation of Cyclosporin A was prepared in the form of a 1%solution in olive oil.

[0171] 2. Determination of the Tolerance for the Cyclic Undecapeptide ofFormula (XV)

[0172] Ocular tolerance was determined according to two methods, namelyaccording to the modified Draize test and using a confocal laserscanning opthalmoscope.

[0173] 2.1 Modified Draize Test (Acute Tolerance Test)

[0174] This evaluation was performed on six male albino rabbits. Foreach animal, one eye received an installation of 50 μl of the solutionas described above, the other eye, not treated, playing the role ofcontrol.

[0175] The clinical evaluation of a possible irritation was performedvisually by evaluating the ocular discharge, the conjuctival chemosisand the conjuctival redness according to the classification described inTable 4 below: TABLE 4 Ocular Normal 0 discharge Slight discharge 1Severe discharge 2 covering a small surface of the cornea Severedischarge 3 covering a large part of the cornea Chemosis Normal 0 Slightchemosis 1 including the nictating 2 membrane Severe chemosis with the 3eye partially closed Severe chemosis with the eye closed Redness Normalblood vessels 0 Some vessels hyperemic 1 Diffuse redness, 2 individualvessels not easily discernible Considerable diffuse 3

[0176] The possible irritation was observed, in each animal, accordingto the rules above, at given intervals spread out over 48 hours afterinstallation, and a total irritation index (I_(irr)) was calculated fromthe total sum of the estimated indices. The results obtained are givenin Table 4 in point 2.3 below.

[0177] 2.2. Confocal Laser Scanning Opthalmoscope (Test to EvaluateSubacute Toxicity Over 4 Days of Administration)

[0178] This test was carried out on the same type of animal aspreviously. 25 μl of a solution as described above were instilled ontothe cornea of the right eye three times a day for four days, and thenonce on the fourth day just before observation. After the finalinstallation, the rabbits were sedated by administering ketaminehydrochloride and xylazine. A total of 25 μl of a solution of sodiumfluorescein at 0.5% by weight/volume were instilled onto the eye inorder to allow selective labeling of the surfaces possibly damaged. Theeye was then rinsed for one minute with a saline solution at 37° C.

[0179] Finally, the eye was observed with a confocal laser scanningopthalmoscope according to the method described by Furrer et al., J.Ocular Pharmacol., 1997, 13, 559. The opthalmoscope was coupled to animage analysis system in order to be able to reconstitute an image inthree dimensions and to allow evaluation of the damaged areas.

[0180] The degree of tolerance is evaluated as a function of thepercentage of corneal lesions and according to the following rule:

[0181] from 0% to 25%: good tolerance;

[0182] from 25% to 40%: acceptance tolerance;

[0183] from 40% to 60%: low tolerance; and

[0184] above 60%: unacceptable tolerance.

[0185] It is noted that it is generally acknowledged that a percentageof lesions less than or equal to 5% corresponds to a usual cellmortality rate in a normal body not subjected to any treatment.

[0186] The results obtained are given in Table 5 in point 2.3 below.

[0187] 2.3. Results of the Ocular Tolerance for the Cyclic Undecapeptideof Formula (XV) TABLE 5 Undecapeptide (XV) Cyclosporin A Draize, I_(irr)1.8 1.9 CLSO, % lesions 7 23

[0188] From Table 5, it appears that a total irritation index 1.8 wasobtained in the Draize test for the cyclic undecapeptide of the formula(XV) and that 7% of the cornea was damaged by administration of thisproduct. These two results demonstrate very good tolerance to the cyclicundecapeptide, this tolerance being clearly improved compared to thatobtained when Cyclosporin A is administered in olive oil.

[0189] For obvious reasons, the subjective improvement in the visualcomfort by using an aqueous solution rather than an oily solution wasnot evaluated on the animal.

[0190] 3. Stability of the Cyclic Undecapeptide of the Formula (XV)

[0191] Samples of the solutions described above were conserved,respectively, at 4° C. and 20° C. Analyses by HPLC were carried outregularly for 3 months. These samples were found to have good stabilityunder such conditions.

[0192] 4. Ex vivo Conversion Kinetics for the Cyclic Undecapeptide ofFormula (XV)

[0193] This conversion kinetics test was carried out by incubating, at37° C., 25 μl of a sample of the solution described above, with gentlestirring, with 8 μl of fresh rabbit tear. 2 μl samples were taken atintervals of 1, 2, 3 and 30 minutes, and were then analyzed by HPLC.

[0194] The results obtained showed that the cyclic undecapeptide offormula (XV) plays its pro-drug role from the first minute of contactwith the rabbit tear, releasing the Cyclosporin A. At 3 minutes, 3% ofthe pro-drug has been converted, and then 4.7% after 30 minutes.

[0195] 5. In vivo Conversion Kinetics for the Cyclic Undecapeptide ofthe Formula (XV)

[0196] This in vivo conversion kinetics test was carried out byinstilling a 25 μl sample of the solution described above, in the righteye of male albino rabbits (4 kg). Tear samples were taken at intervalsof 1, 2, 3, 4 and 20 minutes, and were then analyzed by HPLC.

[0197] The results obtained have been given in FIG. 3. As can be noted,this test confirms the results already obtained in the ex vivoexperiment. The cyclic undecapeptide of formula (XV) (squares) clearlyplays its pro-drug role from the first minute of contact with the rabbittear, releasing the Cyclosporin A (circles), and this release continuesover the following 20 minutes. At 1 minute, the concentration ofCyclosporin A in the tear is 0.025 mg/ml.

6. Conclusions

[0198] From these results, it results that the pro-drugs of the presentinvention offer, for topical administration to the eye, the followingadvantages:

[0199] ease of preparation of a pharmaceutical formulation such as aneye wash by simple dissolution in an aqueous solution without the needfor using oily adjuvants;

[0200] good acute tolerance and very sub acute tolerance, greater thanthose obtained with a pharmaceutical formulation of cyclosporin in oilyform;

[0201] good stability; and

[0202] half-life time suitable for ophthalmic application.

1. A pro-drug consisting of a cyclic undecapeptide in which the peptidechain comprises at least one amino acid residue of general formula (I)below:

in which: the carbon atom C^(a) constitutes one of the links of theundecapeptide ring; the substituents Y each represent a hydrogen atom ortogether constitute a bond; the substituents R¹ and R³ represent,independently of one another, a hydrogen atom, an aralkyl group, analkaryl group, a heteroalkyl group, a heterocyclic group, analkylheterocyclic group, a heterocyclic alkyl group or a linear orbranched alkyl group having from 1 to 6 carbon atoms, optionallysubstituted with at least one of the groups chosen from —COOH, —CONHR⁸,—NHC═NH(NH₂), —NHC═NR⁸ (NH₂), —NH₂, —NHR⁸, —NR⁸ ₂, —N⁺R⁸ ₃, —OH,—OPO(OR⁸)₂, —OPO(OH)(OR⁸), —OPO(OH)₂, —OSO(OR⁸)₂, —OSO(OH)(OR⁸),—OSO(OH)₂, and the various salified forms of these groups, each of thesubstituents R⁸ representing, independently of one another, a linear orbranched alkyl group having from 1 to 6 carbon atoms; the substituentsR² and R⁴ represent, independently of one another a hydrogen atom, analkaryl group, or a linear or branched alkyl group having from 1 to 6carbon atoms; the substituents R⁵ and R⁶ represent, independently of oneanother, a hydrogen atom, an aralkyl group, or a linear or branchedalkyl group having from 1 to 6 carbon atoms; and the substituent R⁷represents an aralkyl group, an alkaryl group, a heteroalkyl group, aheterocyclic group, an alkylheterocyclic group, a heterocyclicalkylgroup or a linear or branched alkyl group having from 1 to 6 carbonatoms, said groups being optionally substituted with at least one of thegroups chosen from —COOH, —CONHR⁸, —NHC═NH(NH₂), —NHC═NR⁸ (NH₂), —NH₂,—NHR⁸, —NR⁸ ₂, —N⁺R⁸ ₃, —OH, —OPO(OR⁸)₂, —OPO(OH)(OR⁸), —OPO(OH)₂,—OSO(OR⁸)₂, —OSO(OH)(OR⁸), —OSO(OH)₂, and the various salified forms ofthese groups, each of the substituents R⁸ having the definition above.2. The pro-drug as claimed in claim 1, characterized in that, in generalformula (I) defining said amino acid residue, at least one of thesubstituents R¹ and R³ represents an aralkyl group, an alkaryl group, aheteroalkyl group, a heterocyclic group, an alkylheterocyclic group, aheterocyclic alkyl group or a linear or branched alkyl group having from1 to 6 carbon atoms, each of said groups being substituted with at leastone of the groups chosen from —COOH, —CONHR⁸, —NHC═NH(NH₂),—NHC═NR⁸(NH₂), —NH₂, —NHR⁸, —NR⁸ ₂, —N⁺R⁸ ₃, —OH, —OPO(OR⁸)₂,—OPO(OH)(OR⁸), —OPO(OH)₂, —OSO(OR⁸)₂, —OSO(OH)(OR⁸), —OSO(OH)₂, and thevarious salified forms of these groups, each of the substituents R⁸having the definition above.
 3. The pro-drug as claimed in claim 2,characterized in that said aralkyl, alkaryl, heteroalkyl, heterocyclic,alkylheterocyclic, heterocyclicalkyl or alkyl groups are substitutedwith at least one of the groups chosen from —NR⁸ ₂, —N⁺R⁸ ₃, —OPO(OH)₂or the various salified forms of these groups, each of the substituentsR⁸ having the definition above.
 4. The pro-drug as claimed in claim 3,characterized in that at least one of said substituents R¹ and R³represents a linear alkyl group having from 1 to 6 carbon atomssubstituted with at least one of the groups chosen from —NR⁸ ₂, —N⁺R⁸ ₃,—OPO(OH)₂ or the various salified forms of these groups, each of thesubstituents R⁸ having the definition above.
 5. The pro-drug as claimedin claim 1, characterized in that, in general formula (I) defining saidamino acid residue, the substituents R5 and R6 cannot simultaneouslyrepresent a hydrogen atom.
 6. The pro-drug as claimed in claim 1,characterized in that, in general formula (I) defining said amino acidresidue, at least one of said substituents R⁵ and R⁶ represents a linearor branched alkyl group having from 1 to 6 carbon atoms, and thesubstituent R⁷ represents an aralkyl group or a linear or branched alkylgroup having from 1 to 6 carbon atoms.
 7. The pro-drug as claimed inclaim 6, characterized in that said substituents R⁵ and R⁶ represent,independently of one another, a hydrogen atom or a methyl group.
 8. Thepro-drug as claimed in claim 1, characterized in that said peptide chaincomprises a single amino acid residue of general formula (I) forming anundecapeptide ring with a linear sequence of ten amino acids of generalformula (II) below: -T-U-V-W-MeLeu-Ala-X-MeLeu-Z-MeVal-  (II) in which:T is chosen from the amino acids Ala, Abu, Nval, Val and Thr; U ischosen from the amino acids Sar, (D)MeSer, (D)MeAla and (D)MeSer(OCOR⁹),with R⁹ representing a hydrogen atom, an alkaryl group, or a linear orbranched alkyl group having from 1 to 6 carbon atoms; V represents anamino acid of general formula (N—R¹⁰)aa, aa being chosen from the aminoacids Val, Leu, Ile, Thr, Phe, Tyr and Thr, and R¹⁰ being a linear orbranched alkyl group having from 1 to 6 carbon atoms; W is chosen fromthe amino acids Val, Nval and Leu; X is chosen from the amino acids(D)Ala, (D)Ser, (D)Hiv, (D)Val and (D)Thr; and Z is chosen from theamino acids Leu and MeLeu.
 9. The pro-drug as claimed in claim 8,characterized in that said linear sequence of ten amino acids is chosenfrom the following sequences of formulae (III) to (XV):-Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (III);-Abu-(D)MeAla-EtVal-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (IV);-Thr-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (V);-Val-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (VI);-Nval-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (VII);-Val-(D)MeAla-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVal-  (VIII);-Val-Sar-MeLeu-Val-MeLeu-Ala-(D)Val-MeLeu-Leu-MeVal-  (IX);-Val-Sar-MeLeu-Val-MeLeu-Ala-(D)Thr-MeLeu-Leu-MeVal-  (X);-Abu-(D)MeSer(OAc)-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-Leu-MeVal-  (XI);-Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ser-MeLeu-MeLeu-MeVal-  (XII);-Thr-Sar-MeLeu-Leu-MeLeu-Ala-(D)-Hiv-MeLeu-Leu-MeVal-  (XIII);and -Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)-Val-MeLeu-Leu-MeVal-  (XIV). 10.The pro-drug as claimed in any one of the preceding claims, having,respectively, formulae (XV) and (XVI) below:


11. The use of a pro-drug as claimed in claim 1, as a medicinal product.12. The use of a pro-drug as claimed in claim 1, for preparing amedicinal product intended for the treatment of pathological conditionsof the mucous membranes.
 13. The use of a pro-drug as claimed in claim1, for preparing a medicinal product intended for the local treatment ofpathological conditions of the eye.
 14. The use as claimed in claim 13,characterized in that said pathological conditions are chosen from drykeratoconjunctivitis, Sjögren's syndrome, forms of allergickeratoconjunctivitis, in particular those resistant to corticosteroids,conjunctivitis producing mucous and synechia, herpetic stromalkeratitis, immune-related limbic keratitis and Thygeson's keratitis, andprevention of corneal transplant rejection, and as an adjuvant treatmentfor filtering surgery.
 15. The use of a pro-drug as claimed in claim 1,for preparing a medicinal product intended to allow prolonged survivalof organ allografts.
 16. The use as claimed in claim 15, characterizedin that said organs bearing a graft are chosen from the kidney, heart,liver, pancreas, lung, small intestine and bone marrow.
 17. The use of apro-drug as claimed in claim 1, for preparing a medicinal productintended to inhibit replication of the human immunodeficiency virus typeI (HIV-1).